Like human cells, budding yeast (S. cerevisiae) contains multiple MAPK cascades. The mating pheromone response pathway (Fus3 MAPK), initiated by a GPCR, is arguably the best understood MAPK pathway in any eukaryote. Also well studied are the high-osmolarity glycerol (HOG) pathway for coping with hyperosmotic stress (Hog1 MAPK), the filamentous growth response triggered by nutrient limitation (Kss1 MAPK), and the cell wall integrity pathway that coordinates cell wall synthesis and repair with cell membrane expansion (Mpk1 MAPK). However, many basic questions remain about how such pathways are arranged to maintain specificity, how such pathways are integrated, and how they modulate the processes and behaviors under their control, especially changes in cell growth and polarity. The overall goal of this project is to use the experimental advantages of yeast to continue to examine fundamental properties of the organization, fidelity, regulation, and function of MAPK signaling pathways, as a means of undercovering additional new principles and processes generally applicable to the highly homologous MAPK pathways in human cells. MAPK- mediated signaling evokes an elaborate network of interlocking events, rather than a simple linear pathway; but, it is not well understood how changes in metabolism, gene expression, and biosynthesis (especially membrane lipid synthesis) are properly coordinated in space and time to achieve dramatic changes in cell morphology. Moreover, certain temporal and spatial aspects of MAPK signaling are imposed by negative feedback mechanisms, and others by the cell cycle machinery, but much more needs to be learned about signal propagation and the mechanisms that modulate the efficiency and duration of signaling events. In particular, pheromone response, filamentous growth, and the HOG pathway share the same MAPKKK (Ste11), but are coupled to different upstream inputs, elicit the appropriate response upon the correct stimulus, yet avoid adventitious activation of the wrong output. How different extracellular signals impinge on the same MAPK elements, yet are deciphered differently, is not fully understood in any organism. To address many of these issues experimentally, our specific aims and goals include: (1) mutational and structural analysis of DEP domain-mediated GPCR recognition; (2) genetic and biochemical studies of the mechanism of MAPK-induced anisotropy in plasma membrane phosphoinositide distribution; (3) genetic and biochemical studies of the control of the remodeling of other plasma membrane lipids in pheromone- and nutrient limitation-induced polarized growth; (4) biochemical and genetic analysis of the mechanism of MAPK-mediated control of the organization and dynamics of the septin filament cytoskeleton; and, (5) determination of the molecular basis by which the stress-activated Hog1 MAPK blocks inappropriate activation of the other two pathways (Fus3 MAPK and Kss1 MAPK) that utilize the same MAPKKK (Ste11). PUBLIC HEALTH RELEVANCE: This proposed project has substantial public health relevance because the growth of many human tumor cells can be traced to mutations that lead directly to inappropriate and persistent MAPK activation. Thus, further elucidation of the fundamental aspects of MAPK signaling may provide new insights for the development of novel and more effective anti-cancer therapies to ameliorate certain prevalent malignancies in people.